,, 董炜灵, 宋阿琳, 李艳玲, 卢玉秋, 王恩召, 刘雄舵, 王萌, 范分良,中国农业科学院农业资源与农业区划研究所/农业农村部植物营养与肥料重点实验室,北京 100081Effects of Straw Addition on Soil Biological N2-Fixation Rate and Diazotroph Community Properties
LI Xu,, DONG WeiLing, SONG ALin, LI YanLing, LU YuQiu, WANG EnZhao, LIU XiongDuo, WANG Meng, FAN FenLiang,Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences/Key Laboratory of Plant Nutrition and Fertilizer, Ministry of Agriculture and Rural Affairs, Beijing 100081通讯作者:
责任编辑: 李云霞
收稿日期:2020-06-21接受日期:2020-12-16网络出版日期:2021-03-01
| 基金资助: |
Received:2020-06-21Accepted:2020-12-16Online:2021-03-01
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李旭, 董炜灵, 宋阿琳, 李艳玲, 卢玉秋, 王恩召, 刘雄舵, 王萌, 范分良. 秸秆添加量对土壤生物固氮速率和固氮菌群落特征的影响[J]. 中国农业科学, 2021, 54(5): 980-991 doi:10.3864/j.issn.0578-1752.2021.05.010
LI Xu, DONG WeiLing, SONG ALin, LI YanLing, LU YuQiu, WANG EnZhao, LIU XiongDuo, WANG Meng, FAN FenLiang.
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0 引言
【研究意义】生物固氮通过少数固氮原核微生物体内的固氮酶系统,将大气中的N2催化转化为植物可利用的形式,每年可为农田生态系统提供氮素输入40—70 Tg N,是农业生态系统中氮素的重要来源之一[1,2,3,4]。生物固氮分为共生固氮和非共生固氮。据报道,作物生物量中约有24%的氮素来自非共生固氮[5,6]。近年来,过度施用氮肥导致的一系列环境问题和不断增加的经济成本都要求我们尽可能提高生物固氮的潜力[7,8,9]。秸秆还田后可补充大量有机碳源和能量,改善土壤理化性质并提高土壤细菌和非共生固氮微生物的数量和活性[10,11],从而促进生物固氮[12,13]。因此,充分利用秸秆资源,发挥非共生固氮微生物的固氮潜力对农田生态系统氮素输入及农业生产具有重要意义。【前人研究进展】非共生固氮微生物的群落组成及其固氮酶活性受到土壤水分和温度,土壤质地及pH,微量元素,养分有效性（N、P）和碳源可利用性等多种土壤和环境因子的综合影响[14,15,16,17,18]。由这些因素引起的固氮微生物群落多样性及结构的变化,可较好地反应其生物固氮的能力[15,19-20]。一些研究表明土壤中固氮酶活性与可利用的植物残茬量和分解率成正相关[21,22,23],作物秸秆还田后可以改变固氮群落结构的组成,增加nifH基因丰度并促进生物固氮[14,20,24-27],但在一些研究中这种关系还不明确[28,29]。此外,有研究发现固氮速率显示出对固氮微生物多样性增加的饱和趋势[15]。有研究表明,在作物秸秆投入的情况下,非共生固氮微生物的固氮潜力可在2—4周的短时间内达到1—12 kg N·hm-2[21,30];另一项测量澳大利亚非共生固氮潜力的研究表明,非共生固氮对作物生长的贡献可能小于10 kg N·hm-2·a-1,但在土壤氮含量较低且碳源充足的情况下,固氮效率可能会更高,在理想（温暖,潮湿）条件下新鲜秸秆投入,或可使固氮潜力高达30—38 kg N·hm-2·a-1 [11,31]。目前关于秸秆投入对非共生固氮影响的研究说法不一,可能是由两方面原因造成：秸秆还田量不同,已有的研究秸秆还田多为全量添加或非限制性的[11,12,13,14,15,16,17,18,19,20,21],例如ROPER在早期田间试验中,秸秆还田量为4.5 t·hm-2,测得固氮速率约为1.1 kg N·hm-2·a-1,关于不同秸秆添加量与固氮微生物固氮速率的定量研究少之又少;关于非共生固氮微生物的固氮能力的研究,绝大多研究采用乙炔还原法（ARA）间接测定,但可能会造成固氮速率的错误估计,由于测定条件的影响,乙炔还原法（ARA）中C2H2﹕N2的比值可能明显偏离理论转换因子,须使用15N2方法对其进行校正[32],15N2方法作为定量生物固氮潜力的唯一直接方法,有着良好的灵敏度和精确度[33]。以往的研究表明,变形杆菌（Proteobacteria）和蓝细菌（Cyanobacteria）是土壤中丰富的固氮微生物[34];同时参与分解玉米秸秆和固氮过程的包括根瘤菌（Rhizobium）、中华根瘤菌（Sinorhizobium）及部分未培养的固氮微生物[35]。受限于多数固氮菌难以培养及早期克隆测序不能全面了解固氮菌群落特征,秸秆投入如何影响固氮菌群落特征仍需进一步探索。【本研究切入点】上述研究表明,土壤固氮微生物对秸秆投入数量的响应有差异,同时测定方法也存在局限。近年来,随着我国“减肥增效”政策的推进,秸秆还田引起人们的广泛关注,在促进生物固氮能力方面,我们对于秸秆还田后如何影响固氮微生物群落及其固氮酶活性仍缺乏清晰地认识,同时由于多数固氮微生物难以培养,使用15N2同化方法,高通量测序等方法对秸秆还田促进生物固氮能力进行研究势在必行。【拟解决的关键问题】采用15N2方法,揭示秸秆添加量与土壤固氮速率和固氮菌群落特征的关系,以期为秸秆还田促进农业生产提供参考。1 材料与方法
1.1 试验设计
供试土样取样自河南省洛阳市孟津县旱地耕层土（0—20 cm）（北纬N34°52′55.74″,东经E112.24′14.98″）,土壤类型为潮土,其基础理化性质为：土壤pH为6.89,土壤有机质36.95 g·kg-1,铵态氮含量8.80 mg·kg-1,硝态氮含量11.00 mg·kg-1,速效磷28.47 mg·kg-1,有效钾210 mg·kg-1,土壤过2 mm筛,去除植物残根和石块等杂质,混匀备用。土壤预培养（25℃,黑暗条件）7 d后,准确称取5.0 g干土等重的鲜土于20 mL灭菌螺口玻璃瓶中,除对照（C0：0）外分别添加粉碎玉米秸秆（C1：0.2 mg·g-1;C2：1.0 mg·g-1;C3：2.0 mg·g-1;C4：4.0 mg·g-1;C5：10.0 mg·g-1）,混合均匀后,加入1.20 mL无菌水平衡水分至60%田间持水量,随后加橡胶塞用铝盖旋紧并检查气密性。15N2处理使用真空泵将玻璃瓶抽成真空,用纯O2反复置换3次以保证瓶中不含N2,随后瓶内气体被置换为15N2﹕O2（v/v= 4﹕1）,每7天按照上述方法重新置换气体,为避免可能的污染和对固氮速率的高估[36],15N2气体使用前均采用OHYAMA等[37]的方法进行清洗后使用。采用实验室空气作为对照,每7天敞口曝气30 min,所有处理黑暗条件下25℃培养28 d。
1.2 样品采集及指标测定
在培养第28天对样品进行破坏性取样,土壤取出后混合均匀,一部分4℃保存,用于理化性质及固氮速率测定,待测完无机氮后,风干磨细用于其他土壤理化指标的测定,其中NO3--N和NH4+-N使用0.01 mol·L-1 CaCl2浸提,后续比色测定;土壤速效磷用0.5 mol·L-1 NaHCO3浸提,钼锑抗比色法测定;土壤速效钾用1 mol·L-1醋酸铵浸提,原子吸收仪测定;土壤pH用快速pH测定仪测定;土壤有机质采用K2Cr2O7氧化外加热法;称取少量15N2处理土壤,置于烘箱70℃烘干72 h,随后研磨过100目筛后送样,使用元素分析仪串联同位素质谱仪（IsoPrime100）测定土壤样品中的15N丰度。然后,通过比较15N2处理的土壤相对于对照原子丰度的差异,计算生物固氮（biological N2 fixation）,计算公式如下：BNF（μg·g-1DW）= total Nsample（μg·g-1 DW）×atom% 15Nexcess
式中,BNF（μg·g-1DW）为生物固氮量;atom% 15Nexcess=atom%15Nsample-atom%15Ncontrol;total Nsample 为总氮含量。另一部分存于-80℃,用于土壤DNA提取。
1.3 DNA提取、PCR扩增和测序
使用FastDNA SPIN Kit for Soil 试剂盒（MP Biomedicals,USA）提取土壤总DNA,随后使用1%琼脂糖凝胶电泳检验DNA提取质量,用NanoDrop 2000微量分光光度计（Thermo Scientific,USA）检测其浓度和纯度,保存至-20℃备用。将提取的DNA原液稀释至约10 ng·μL-1作为PCR扩增模板,采用two-step PCR法进行扩增。第一轮扩增引物为带有16 bp头部序列的polF（TGC GAY CCS AAR GCB GAC TC）,polR（ATS GCC ATC ATY TCR CCG GA）,扩增体系为25 μL,包含：2.5 μL 10×buffer,2 μL dNTP,0.25 μL rTaq（TaKaRa）、10 μmol·L-1的前后引物各1 μL、1 μL模板DNA和17.25 μL ddH2O,每个样品3个重复。第一轮PCR扩增程序：95℃预变性3 min后,95℃变性30 s,62℃退火30 s,72℃延伸60 s,循环40次,循环结束后72℃保持10 min,4℃条件下结束。将同一样本的3次重复的DNA扩增混匀后,用1%的琼脂糖凝胶电泳检测。第二轮扩增使用第一轮扩增产物作为模板DNA,前引物为长度为16 bp的不同barcode引物,后引物为polR（ATS GCC ATC ATY TCR CCG GA）,扩增体系为50 μL,包括：5 μL 10×buffer,4 μL dNTP,0.5 μL rTaq（Takara）、10 μmol·L-1的前后引物各2 μL、2 μL模板DNA和34.5 μL ddH2O,每个样品3次重复,扩增程序循环次数为10次,其他设定同第一轮。扩增引物及策略参照GABY等[38,39]的方法。
将同一样本的3次重复的DNA扩增混匀后,用1%的琼脂糖凝胶电泳检测。使用PicoGreen试剂盒测定所得PCR产物浓度,等摩尔量混匀后,采用DNA纯化试剂盒（TIANGEN Biotech,Beijing,China）进行纯化回收,按照每个样本的测序量要求,进行相应比例的混合。混合后的样品通过Illumina Hiseq2500 PE250平台（Novogene公司）进行nifH序列测定,测序数据已上传至GSA数据库（GSA编号：CRA002883）。
1.4 绝对定量PCR（qPCR）
qPCR采用ABI 7900实时定量PCR系统进行检测,选用的荧光试剂是SYBR Premix Ex TaqTM Ⅱ。qPCR的标准曲线制作方法如下：以对照组土样提取DNA为模板进行PCR扩增,扩增引物是ploF（TGC GAY CCS AAR GCB GAC TC）和polR（ATS GCC ATC ATY TCR CCG GA）,体系如上所述,检查PCR产物并使用DNA纯化试剂盒（TIANGEN Biotech,Beijing,China）纯化回收产物;使用pMDTM 18-T Vector Cloning Kit（TaKaRa Bio Inc.）试剂盒将回收纯化后的PCR产物与pMD18载体连接;最后使用MiniBEST Plasmid Purification Kit Ver.4.0（TaKaRa Bio Inc.）质粒纯化试剂盒对菌液进行质粒提取与纯化,使用NanoDrop 2000微量分光光度计（Thermo Scientific,USA）检测其浓度和纯度,计算质粒拷贝数并逐步稀释到108—101拷贝数备用[40,41]。qPCR扩增引物是ploF（TGC GAY CCS AAR GCB GAC TC）和polR（ATS GCC ATC ATY TCR CCG GA）,扩增体系为15 μL,包含7.5 μL 2×SYBR Premix Ex Taq II、0.3 μL 50×ROX Ref+erence Dye、10 μmol·L-1的前后引物各0.6 μL、2 μL DNA模板和4.0 μL ddH2O,每个样品3次重复。扩增程序：95℃预变性30 s,95℃解链5 s,62℃退火30 s,72℃延伸60 s,83℃采集信号10 s,40个循环。1.5 生物信息学分析
高通量测序数据经过数据质控,进行序列拼接、过滤并去除嵌合体后得到高质量序列。随后以97%相似性水平进行分类操作单元（OTU）聚类,使用nifH数据库进行物种分类注释及后续分析[42]。1.6 数据处理
试验数据采用Excel 2010和SPSS 21软件进行统计分析,利用ANOVA进行单因素方差分析,利用Duncan法进行多重比较（P<0.05）。土壤理化性质与固氮群落指标及其功能菌属spearman相关关系采用R中的“psych”包分析;群落结构差异及环境因子对群落结构影响的主坐标（PCoA）和冗余分析（RDA）由R中的“vegan”包完成;偏最小二乘路径分析建模（PLS-PM）由R中的“plspm”分析完成。作图软件为Microsoft Excel及R中“pheatmap”及“ggplot2”包。2 结果
2.1 不同秸秆添加量对土壤理化性质及生物固氮的影响
由表1可知,秸秆添加培养后,随着秸秆添加量增加,土壤pH及NO3--N含量均呈下降趋势,与C0相比,土壤NO3--N含量显著下降,土壤的NH4+-N含量各处理之间无显著差异。利用15N2同化方法对固氮速率定量以衡量固氮酶活性,不同秸秆添加量处理的固氮微生物的固氮速率,相比于不添加秸秆处理（C0）,处理C2、C3、C4及C5土壤潜在固氮能力显著增加（P<0.005）。随着秸秆添加量的增加,C3、C4及C5处理之间固氮速率无显著性差异,在培养期间（28 d）潜在生物固氮可达到2.71—3.05 μg N·g-1DW,约为0.10—0.11 mg N·kg -1DW·d-1,相比C0固氮速率提高了35.5%—52.5%。Table 1
表1
表1不同处理的土壤理化性质及生物固氮
Table 1
| 处理 Treatment | pH | NO3--N (mg·kg-1) | NH4+-N (mg·kg-1) | TN (g·kg-1) | 生物固氮量 Biological N2-fixation (μg N·g-1DW) |
|---|---|---|---|---|---|
| C0 | 7.07±0.01ab | 15.33±0.26a | 11.81±1.07a | 1.18±0.01e | 2.00±0.18d |
| C1 | 7.10±0.01a | 14.44±0.14b | 11.98±0.93a | 1.21±0.00cd | 2.25±0.05cd |
| C2 | 7.03±0.02b | 13.80±0.09c | 11.53±0.77a | 1.20±0.01de | 2.50±0.11bc |
| C3 | 7.06±0.03ab | 11.25±0.14d | 11.77±0.66a | 1.24±0.01b | 2.78±0.10ab |
| C4 | 6.96±0.03c | 5.97±0.30e | 11.13±0.14a | 1.23±0.00bc | 2.71±0.15ab |
| C5 | 6.93±0.03c | 3.46±0.24f | 11.85±0.81a | 1.28±0.00a | 3.05±0.13a |
新窗口打开|下载CSV
2.2 固氮微生物群落多样性及群落组成
图1-A为基于97%相似度的OTU数据,采用加权（Weighted Unifrac）算法分别对不同秸秆添加量的微生物群落结构的主成分分析结果。其中,第一主成分和第二主成分的方差贡献率分别为10.97%和8.96%,累计方差贡献率为19.93%;在第一轴上,C0、C1与C4、C5在第一轴上能够很好地分开,解释量为10.97%。图1
新窗口打开|下载原图ZIP|生成PPT图1不同秸秆添加量土壤固氮微生物群落结构差异的PCoA分析（A）,nifH基因拷贝数（B）及其香农-威尔指数（C）,门水平下相对丰度（D）及优势功能菌属相对丰度（>1%）（E）
不同字母表示不同秸秆添加量处理差异达到显著水平（P<0.05）,*表示P<0.05
Fig. 1Diazotroph community structure assessed by principal coordinate analysis (A), nifH gene copy number (B) and Shannon-Wiener index (C) of soil diazotroph (>1%); Relative abundance of phylum (%) (D), relative abundance (%) of most abundant genera (>1%) (E) under different straw additions
Values followed by different letters means significant differences in different straw additions (P<0.05), * Mean P<0.05
图1-B为15N2处理对应不同秸秆添加量土壤中的固氮微生物nifH基因拷贝数,各处理固氮微生物nifH基因拷贝数为5.48×107—9.20×107 copies/(g soil)。其中,相比于C0,C4及C5处理固氮微生物nifH基因拷贝数均显著提高,显著提高了固氮微生物数量和活性（P<0.05）。当秸秆添加量达到4.0 mg·g-1后,再增加秸秆添加量对土壤固氮微生物nifH基因拷贝数无显著影响。图1-C为固氮微生物群落结构香农-威尔指数多样性分析结果,香农-威尔指数越高,微生物群落多样性越丰富,反之亦然。其中,C4、C5水平的香农-威尔指数显著低于其他4个处理（P<0.05）,其他4个处理多样性无显著差异。
图1-D为不同秸秆添加量下门水平下固氮微生物群落结构相对丰度,不同处理固氮微生物群落组成主要为变形杆菌（Proteobacteria）、蓝细菌（Cyanobacteria）、厚壁菌（Firmicutes）、放线菌（Actinobacteria）和螺旋体细菌（Spirochaetes）。随着秸秆添加量的增加,C2、C3、C4和C5相比C0水平蓝细菌的相对丰度显著增加（P<0.05）,但当秸秆添加量达到10.0 mg·g-1时,其相对丰度反而有所降低。对于变形杆菌,C3和C4水平相较于C0、C1、C5水平其相对丰度显著减少（P<0.05）。对于厚壁菌,C4相较于C0、C1、C2水平的相对丰度下降极为显著（P<0.01）,C5水平相对于C0、C1、C2和C3水平其相对丰度下降极为显著（P<0.01）。对于放线菌,C5相较于C0、C1、C2和C3水平显著减少（P<0.05）。对于螺旋体门细菌,C2、C3、C4和C5相较于C0水平下相对丰度显著增加（P<0.05）。
图1-E为属水平下不同秸秆添加量下固氮微生物群落结构相对丰度,包含22个优势功能菌属（相对丰度大于1%）,其中包括多种α-型、β-型、γ-型变形菌（Proteobacteria）,以及一种蓝细菌,如贺氏伪枝藻属（Scytonema hofmanni）。慢生根瘤菌（Bradyrhizobium）为最丰富菌属,随着秸秆添加量的增加,C5相较于C0、C1、C2、C3水平相对丰度差异显著（P<0.05）,显著增加了12.07%—14.13%。贺氏伪枝藻属（Scytonema hofmanni）随着秸秆添加量的增加其相对丰度逐渐增加,但当秸秆添加量达C5水平其相对丰度反而降低（P<0.05）。Amorphomonas属和Methylocapsa属的相对丰度随着秸秆添加量逐渐增加,但当秸秆添加量达C4和C5水平其相对丰度相较于C3反而显著降低（P<0.05）。随着秸秆添加量增加,相对丰度成增加趋势的属有Scytonema sp. FGP-7A和Treponema属。此外,中华根瘤菌（Sinorhizobium）和固氮螺菌（Azospirillum）,在秸秆添加水平达到C5水平其相对丰度才显著增加（P<0.05）。随着秸秆添加量的增加,相对丰度呈下降趋势的属有弧菌属（Vibrio）、硫红螺旋菌属（Thiorhodospira）、弗兰克菌属（Frankia）、太阳杆菌属（Heliobacterium）及Heliorestis属。
2.3 相关性分析
表2为环境因子与土壤固氮微生物群落的Spearman相关性分析。相关性分析表明,nifH基因拷贝数与秸秆添加量（r=0.708,P<0.005）呈显著正相关,但与pH（r=-0.623,P<0.005）和NO3--N含量（r=-0.737,P<0.005）呈显著负相关,其中,NO3--N含量与nifH基因拷贝数相关性最强。固氮速率与秸秆添加（r= 0.804,P<0.005）和TN（r=0.598,P<0.005）呈显著正相关,与NO3--N含量（r=-0.828,P<0.005）呈显著负相关,其中,NO3--N含量与固氮速率相关性最强。Table 2
表2
表2环境因子与土壤固氮微生物丰度、多样性和固氮速率的相关性分析
Table 2
| nifH基因拷贝数 nifH copy numbers | 固氮速率 N2-fixation rate | 香农-威尔指数 Shannon-Wiener index | ||||
|---|---|---|---|---|---|---|
| R | P | R | P | R | P | |
| 秸秆添加Straw addition | 0.708 | 0.000 | 0.804 | 0.000 | -0.634 | 0.002 |
| NO3--N | -0.737 | 0.000 | -0.828 | 0.000 | 0.621 | 0.002 |
| NH4+-N | -0.166 | 0.508 | 0.019 | 0.929 | 0.054 | 0.851 |
| TN | 0.483 | 0.027 | 0.598 | 0.003 | -0.340 | 0.144 |
| pH | -0.623 | 0.002 | -0.410 | 0.073 | 0.678 | 0.001 |
新窗口打开|下载CSV
图2-A为群落组成结构同环境因子的冗余分析（RDA）结果表明,所检测的环境因子对土壤固氮微生物群落结构差异解释量为35.54%,RDA1和RDA2解释量分别为28.38%和3.67%,生物固氮量（BNF）及土壤环境因子中的NO3--N含量和pH的影响作用达到显著。如图2-B所示,使用PLS-PM方法来研究不同秸秆添加量（straw addition）、培养后土壤pH及有效氮浓度（NO3-、NH4+）、nifH基因丰度（nifH abundance）,固氮微生物群落结构（nifH communitystructure PCoA）和生物固氮（BNF）之间的关系。其中,路径系数表示变量之间线性关系（直接效应）的方向和强度,间接效应为预测变量和响应变量之间路径系数相乘,潜在变量之间总效应是二者的加和,拟合优度（GoF）表示模型的质量。分析表明,随着秸秆添加量的增加,秸秆添加与NO3-含量和pH呈显著负相关,其中,与NO3-含量负相关系数最大（-0.93）。NO3-含量与nifH群落结构显著正相关（0.65）,与nifH基因丰度呈负相关（-0.69）,表明秸秆添加后,固氮微生物对土壤NO3-的利用导致了群落结构的显著变。此外,nifH群落结构与BNF显著负相关（-0.87）,表明在nifH群落结构改变后,对BNF造成显著影响。尽管pH在秸秆添加后显著变化且与BNF有着直接的显著相关性,但与间接效应相抵消后,pH对BNF总效应仅为0.43。通过观察各变量之间的总效应,可以发现秸秆添加（0.67）、NO3-（-0.89）及nifH微生物群落结构（-0.87）对生物固氮速率影响较大;秸秆添加（-0.85）及NO3-（-0.70）对nifH微生物群落结构影响较大。
图2
新窗口打开|下载原图ZIP|生成PPT图2土壤固氮微生物群落结构差异与环境因子的RDA分析（A）及PLS-PM分析（B）
***,**,*分别表示P<0.001,P<0.01,P<0.05。
Fig. 2Redundancy analysis (A) of the diazotroph community structure affected by physicochemical variables and PLS-PM analysis (B)
***, **, * mean P<0.001, P<0.01, P<0.05 respectively. The same as
图3为固氮微生物优势功能菌属与土壤理化性状及生物固氮速率的Spearman相关性分析。分析表明,属水平上大多数优势土壤固氮微生物相对丰度与土壤硝态氮及生物固氮量显著相关。其中,土壤中的优势功能菌属（>1%）Bradyrhizobium、Scytonema hofmanni、Scytonema sp. FGP-7A、Azospirillum和Treponema等属与生物固氮速率呈显著正相关,此外,Anabaena variabilis、Ectothiorhodospira等属也与生物固氮速率呈显著正相关,但其相对丰度为较低水平;而Vibrio、Thiorhodospira、Heliobacterium、Anabaena azotica、Methylobacter、Desulfobacteraceae、 Heliorestis、Pseudomonas和Oscillatoria sp. MMG-2等属与生物固氮速率呈显著负相关。NO3--N对菌属相对丰度影响与BNF的趋势相反,且有着较强的相关性,土壤pH的趋势和NO3--N类似,如Bradyrhizobium、Scytonema hofmanni和Scytonema sp. FGP-7A相对丰度与BNF呈显著正相关,而与NO3--N和pH呈显著负相关。
图3
新窗口打开|下载原图ZIP|生成PPT图3土壤固氮微生物功能菌属与土壤理化性状及固氮速率的Spearman相关性分析
Fig. 3Spearman correlation analysis between relative abundance of diazotroph genera and soil physicochemical variables and N2-fixation rate
3 讨论
许多研究表明,秸秆添加后补充的大量有机碳源和能量会导致土壤中微生物群落利用无机态氮,提高土壤中细菌和非共生固氮微生物的数量和活性[10,11],从而促进生物固氮[12,13]。有机物料添加有利于促进nifH基因丰度及固氮功能的维持[43,44,45],但其对固氮微生物的影响可能也与有机物料的数量有关[46]。在本研究中,与C0相比,除C1（0.2 mg·g-1）处理固氮速率增加不显著外,其余处理固氮速率均显著提高,但随着秸秆添加量增加到C3（2.0 mg·g-1）水平后,固氮速率不再显著增加。秸秆中含有纤维素和半纤维素,土壤中只有少数几种固氮微生物（Azospirillum）能够直接利用秸秆进行固氮[47],大多数固氮微生物依赖于其他微生物将其分解为较小的产物。过量秸秆的添加并不代表其满足微生物生长所需的可利用碳源,进而导致固氮速率不再显著增加。相关性分析表明固氮速率与NO3--N含量呈显著负相关（表2）,与秸秆添加量呈显著正相关。此外,固氮速率与TN含量呈显著正相关可能归因于秸秆添加输入的大量有机氮源。碳源的数量是导致微生物群落竞争的重要因子,很大程度上决定了微生物活性和群落结构组成[48]。本研究中,当秸秆添加量达到C4水平后,增加秸秆添加量对nifH基因丰度无显著影响（图1-B）。相关性分析表明,nifH基因丰度与秸秆添加量呈显著正相关,与NO3--N含量呈显著负相关（P<0.001）。以往的研究发现固氮速率显示出对固氮微生物多样性增加的饱和趋势[15]。在本研究中也发现了类似的趋势,当秸秆添加量达到C3水平后,香农-威尔指数显著下降,表明随着秸秆的过量添加,碳源可利用性的限制导致了固氮微生物的群落竞争,群落多样性显著下降。
以往的研究表明参与玉米秸秆分解的微生物主要包括变形杆菌、厚壁菌及放线菌,如Rhizobium、Bradyrhizobium、Heliobacillus和Frankia等[35]。Bradyrhizobium、Methylocapsa及Cyanobacteria是土壤中较为常见的固氮微生物[38]。在本研究中,固氮微生物主要包含多种α-型、β-型、γ-型变形菌（Proteobacteria）,以及蓝细菌（Cyanobacteria）。在属水平上主要由Bradyrhizobium、Sniorhizobium、Vibrio、Rhizobium、Thiorhodospira和Scytonema hofmanni等属组成。其中,Bradyrhizobium可参与秸秆分解且为最丰富菌属,不仅可与豆科植物共生固氮,作为根际细菌也可促进非豆科植物生长[49],随着秸秆添加量的增加,其相对丰度显著增加了12.07%—14.13%（图1-E）。Scytonema hofmanni、Amorphomonas和Methylocapsa等属的相对丰度随着秸秆添加量逐渐增加,但当秸秆添加量一定水平后其相对丰度反而显著降低（P<0.05）。随着秸秆添加量的增加,Vibrio、Thiorhodospira、Frankia和Heliorestis等属相对丰度呈下降趋势。Bradyrhizobium作为优势菌属,与生物固氮速率呈显著正相关（P<0.05）,但其与固氮速率的相关性不如Scytonema hofmanni,这可能与其作为兼性固氮菌,会优先支持自身营养生长而不是用于固氮有关;同时,秸秆添加有增加Ectothiorhodospira相对丰度的趋势,其与生物固氮速率呈显著正相关（P<0.01）,进一步表明碳源的数量对固氮微生物群落结构的影响。此外,Scytonema sp. FGP-7A、Azospirillum和Treponema等属与BNF均呈显著正相关。上述结果表明,碳源的数量是影响土壤固氮微生物群落特征的关键因素。
除了上述分析,本研究还采用了PLS-PM分析和RDA分析方法,结果均表明土壤固氮微生物群落结构的差异与NO3--N含量紧密相关。之前的研究表明,当铵态氮含量较低的情况下投入有机物料,硝态氮的利用更加显著,进而导致土壤NO3--N含量下降[50]。结合Spearman相关性分析结果,说明随着秸秆量的增加,导致土壤中硝态氮的降低,与生物固氮速率呈显著正相关且与硝态氮呈显著负相关的功能菌属诸如Bradyrhizobium、Scytonema hofmanni和Scytonema sp. FGP-7A等菌属的相对丰度显著增加,从而促进了固氮微生物对N2的固定。固氮微生物群落多样性在秸秆添加量达到C3水平后显著下降（图1-C）,生物固氮速率在秸秆添加量达到C3水平后无显著差异（表1）,虽然表明碳源可利用性对生物固氮的制约,但添加秸秆后在培养期间（28 d）潜在固氮总量可达到2.71—3.05 μg N·g-1DW,约为0.10—0.11 mg N·kg-1DW·d-1,相比C0固氮速率提高了35.5%—52.5%。以上结果说明了适量添加秸秆后可显著提高生物固氮潜力并改变固氮菌群落结构特征。
据统计,2011年,我国通过秸秆还田投入有机碳储量平均为0.89 Mg C·hm-2 [51],秸秆资源对促进生物固氮潜力有十分积极的意义。本研究表明,C0处理固氮速率为0.07 mg N·kg -1DW·d-1,若以平均土壤容重1.2 g·cm-3来计算,固氮速率约为63 kg N·hm-2·a-1;C3、C4和C5处理固氮速率约为87—96 kg N·hm-2·a-1。与以往研究不同的是,本研究通过15N2方法进行定量,肯定了非共生固氮在水分和温度适宜的条件下对氮素投入的重要作用;土壤固氮微生物多样性在秸秆添加量达到C3（2.0 mg·g-1）水平后显著下降,与固氮速率显著正相关的部分优势功能菌属相对丰度在秸秆添加量达到C4（4.0 mg·g-1）水平后显著下降。为尽可能提高土壤生物固氮潜力,综合考虑固氮微生物多样性、nifH基因拷贝数及生物固氮速率等条件,在本研究基础上得出最佳秸秆添加水平应为4.0 mg·g-1,其潜在固氮总量约为87 kg N·hm-2·a-1,相比对照提高了38.1%。本研究结果进一步表明,土壤固氮微生物多样性及群落结构对碳源数量具有明显响应,可明显提高土壤固氮潜力,对秸秆还田数量调节土壤固氮微生物群落结构及促进生物固氮具有重要指导意义。然而,由于固氮微生物对环境变化十分敏感,田间条件下秸秆还田对固氮微生物群落及生物固氮的调控作用尚需进一步研究。
4 结论
不同秸秆添加量对土壤固氮菌群落特征具有明显影响,添加秸秆后,土壤固氮微生物nifH基因拷贝数呈增加趋势,土壤固氮微生物多样性在秸秆添加量达到2.0 mg·g-1后显著下降,与固氮速率显著正相关的部分优势功能菌属相对丰度在秸秆添加量达到4.0 mg·g-1后显著下降。相比于不添加秸秆,添加秸秆4.0 mg·g-1后,可增加土壤生物固氮速率约为26 kg N·hm-2·a-1。固氮微生物群落结构因秸秆添加量不同而聚集为不同组别。因此,秸秆添加对固氮微生物多样性和群落结构的调控作用主要受碳源的数量影响。此外,固氮微生物群落结构及优势功能菌属相对丰度与固氮速率及NO3--N含量紧密相关,表明秸秆添加后因其较高的C/N比,使得固氮微生物增加对无机态氮（NO3--N）的利用,显著提高固氮微生物的数量和活性,从而促进生物固氮。此外,秸秆添加后造成土壤NO3--N含量的差异可能是驱动土壤固氮微生物群落组成的重要环境因子。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
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Humans continue to transform the global nitrogen cycle at a record pace, reflecting an increased combustion of fossil fuels, growing demand for nitrogen in agriculture and industry, and pervasive inefficiencies in its use. Much anthropogenic nitrogen is lost to air, water, and land to cause a cascade of environmental and human health problems. Simultaneously, food production in some parts of the world is nitrogen-deficient, highlighting inequities in the distribution of nitrogen-containing fertilizers. Optimizing the need for a key human resource while minimizing its negative consequences requires an integrated interdisciplinary approach and the development of strategies to decrease nitrogen-containing waste.
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DOI:10.1038/s41598-016-0001-8URLPMID:28442746 [本文引用: 1]
Krabbe disease (KD) is a neurodegenerative disorder caused by the lack of beta- galactosylceramidase enzymatic activity and by widespread accumulation of the cytotoxic galactosyl-sphingosine in neuronal, myelinating and endothelial cells. Despite the wide use of Twitcher mice as experimental model for KD, the ultrastructure of this model is partial and mainly addressing peripheral nerves. More details are requested to elucidate the basis of the motor defects, which are the first to appear during KD onset. Here we use transmission electron microscopy (TEM) to focus on the alterations produced by KD in the lower motor system at postnatal day 15 (P15), a nearly asymptomatic stage, and in the juvenile P30 mouse. We find mild effects on motorneuron soma, severe ones on sciatic nerves and very severe effects on nerve terminals and neuromuscular junctions at P30, with peripheral damage being already detectable at P15. Finally, we find that the gastrocnemius muscle undergoes atrophy and structural changes that are independent of denervation at P15. Our data further characterize the ultrastructural analysis of the KD mouse model, and support recent theories of a dying-back mechanism for neuronal degeneration, which is independent of demyelination.
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DOI:10.1371/journal.pbio.1001124URLPMID:21857803 [本文引用: 1]
Globally, we are applying excessive nitrogen (N) fertilizers to our agricultural crops, which ultimately causes nitrogen pollution to our ecosphere. The atmosphere is polluted by N(2)O and NO(x) gases that directly and indirectly increase atmospheric warming and climate change. Nitrogen is also leached from agricultural lands as the water-soluble form NO(3)(-), which increases nutrient overload in rivers, lakes, and oceans, causing
DOI:10.1016/j.soilbio.2015.09.018URL [本文引用: 1]
DOI:10.1126/science.1209090URLPMID:21778391 [本文引用: 1]
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DOI:10.1071/SR05122URL [本文引用: 4]
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通过两年田间裂区设计试验,研究了不同土壤耕作方式(常规耕作、深耕、深松)与秸秆还田(秸秆还田、秸秆不还田)对冬小麦-夏玉米一年两熟农田土壤微生物数量、酶活性和作物产量的影响.结果表明: 深松(耕)和秸秆还田不仅降低了土壤容重,提高了土壤有机碳含量,而且增加了土壤微生物数量、土壤酶活性和作物产量,且二者对夏玉米季的影响大于冬小麦季.与常规耕作+无秸秆还田相比,深耕+秸秆还田、深松+秸秆还田处理的20～30 cm土壤容重分别降低8.5%和6.6%,土壤有机碳含量分别提高14.8%和12.4%,土壤微生物数量、土壤酶活性分别提高45.9%、33.9%和34.1%、25.2%,作物产量分别提高18.0%和19.3%,且两处理间无显著差异.说明土壤深松(耕)结合秸秆还田有利于作物产量、土壤微生物数量和酶活性的提高.
URL [本文引用: 3]
通过两年田间裂区设计试验,研究了不同土壤耕作方式(常规耕作、深耕、深松)与秸秆还田(秸秆还田、秸秆不还田)对冬小麦-夏玉米一年两熟农田土壤微生物数量、酶活性和作物产量的影响.结果表明: 深松(耕)和秸秆还田不仅降低了土壤容重,提高了土壤有机碳含量,而且增加了土壤微生物数量、土壤酶活性和作物产量,且二者对夏玉米季的影响大于冬小麦季.与常规耕作+无秸秆还田相比,深耕+秸秆还田、深松+秸秆还田处理的20～30 cm土壤容重分别降低8.5%和6.6%,土壤有机碳含量分别提高14.8%和12.4%,土壤微生物数量、土壤酶活性分别提高45.9%、33.9%和34.1%、25.2%,作物产量分别提高18.0%和19.3%,且两处理间无显著差异.说明土壤深松(耕)结合秸秆还田有利于作物产量、土壤微生物数量和酶活性的提高.
URL [本文引用: 3]
秸秆还田作为全球有机农业的重要环节, 对维持农田肥力, 减少化肥使用, 提高陆地土壤碳汇能力具有积极作用。秸秆还田主要通过增加土壤有机质和提高氮肥利用率来改善农田生产环境, 获得高农业生产能力。有效的秸秆还田能为土壤中的微生物提供丰富的碳源, 刺激微生物活性, 提高土壤肥力; 同时矿化的秸秆组分能促进土壤氮循环和矿化, 提高氮素有效性。秸秆还田能够促使集约化高氮输入的农田生态系统维持正常的碳氮比例, 减少氮素淋洗损失, 改善土壤结构板结和连作障碍等现象。目前我国农田秸秆还田率不足50%, 与欧美国家高达90%多的秸秆还田率相比, 还具有很大的发展潜力。因此加强我国秸秆还田率能够逐渐改变我国耕地土壤存在的有机质含量和品质下降、氮素损失严重等现象。目前应进一步深入研究秸秆还田对有机质及氮素有效性的影响机制, 并结合长期监测试验, 以及多种秸秆还田技术进行比较研究, 发展适合当地秸秆还田模式, 促进我国农业生态系统的可持续性。
URL [本文引用: 3]
秸秆还田作为全球有机农业的重要环节, 对维持农田肥力, 减少化肥使用, 提高陆地土壤碳汇能力具有积极作用。秸秆还田主要通过增加土壤有机质和提高氮肥利用率来改善农田生产环境, 获得高农业生产能力。有效的秸秆还田能为土壤中的微生物提供丰富的碳源, 刺激微生物活性, 提高土壤肥力; 同时矿化的秸秆组分能促进土壤氮循环和矿化, 提高氮素有效性。秸秆还田能够促使集约化高氮输入的农田生态系统维持正常的碳氮比例, 减少氮素淋洗损失, 改善土壤结构板结和连作障碍等现象。目前我国农田秸秆还田率不足50%, 与欧美国家高达90%多的秸秆还田率相比, 还具有很大的发展潜力。因此加强我国秸秆还田率能够逐渐改变我国耕地土壤存在的有机质含量和品质下降、氮素损失严重等现象。目前应进一步深入研究秸秆还田对有机质及氮素有效性的影响机制, 并结合长期监测试验, 以及多种秸秆还田技术进行比较研究, 发展适合当地秸秆还田模式, 促进我国农业生态系统的可持续性。
DOI:10.1071/SR06022URL [本文引用: 3]
DOI:10.1038/ismej.2008.82URLPMID:18769458 [本文引用: 5]
Microbial ecologists continue to seek a greater understanding of the factors that govern the ecological significance of microbial community structure. Changes in community structure have been shown to have functional significance for processes that are mediated by a narrow spectrum of organisms, such as nitrification and denitrification, but in some cases, functional redundancy in the community seems to buffer microbial ecosystem processes. The functional significance of microbial community structure is frequently obscured by environmental variation and is hard to detect in short-term experiments. We examine the functional significance of free-living diazotrophs in a replicated long-term tillage experiment in which extraneous variation is minimized and N-fixation rates can be related to soil characteristics and diazotroph community structure. Soil characteristics were found to be primarily impacted by tillage management, whereas N-fixation rates and diazotroph community structure were impacted by both biomass management practices and interactions between tillage and biomass management. The data suggest that the variation in diazotroph community structure has a greater impact on N-fixation rates than do soil characteristics at the site. N-fixation rates displayed a saturating response to increases in diazotroph community diversity. These results show that the changes in the community structure of free-living diazotrophs in soils can have ecological significance and suggest that this response is related to a change in community diversity.
URLPMID:19412595 [本文引用: 2]
DOI:10.1128/AEM.03054-09URLPMID:20601513 [本文引用: 2]
A diverse soil microbial community is involved in nitrogen cycling, and these microbes can be affected by land management practices and weed invasion. We surveyed 20 woodlands with a history of livestock grazing, with livestock recently excluded from 10 sites. We investigated whether soil nutrients were lower when grazing was excluded and higher when exotic grasses dominated the understory. Second, using quantitative real-time PCR, we investigated whether microbial nitrogen functional gene (NFG) abundance was altered with soil nutrient enrichment, livestock exclusion, and exotic grass invasion. The target genes were chiA (decomposition-ammonification), nifH (nitrogen fixation), nirK and narG (denitrification), and bacterial amoA (nitrification). Woodland soils were enriched in phosphorus and nitrogen compared to reference condition sites, but soil nutrients were not lower following livestock exclusion. Total nitrogen and nifH were negatively correlated in grazed woodlands, suggesting that aboveground herbivory reduces the capacity for belowground nitrogen fixation. Woodlands dominated by exotic grasses had higher levels of nitrate, narG, and nirK than those dominated by native grasses. We hypothesize that the increase in potential for denitrification was due to increases in soil nitrate, rather than changes in plant composition. Overall, soil physicochemistry explained more variation in NFG abundance than livestock presence or plant invasion, particularly for chiA and bacterial amoA, with significant relationships between the abundance of all five NFGs and total nitrogen or nitrate. All woodlands investigated had a history of anthropogenic disturbance and nutrification, and soil nutrient levels and the abundance of NFGs are likely to be related to long-term land management practices.
DOI:10.1146/annurev-ecolsys-102710-145034URL [本文引用: 2]
URLPMID:20454976 [本文引用: 2]
DOI:10.1016/j.pedobi.2010.08.001URL [本文引用: 3]
Abstract
N2-fixation by free-living (diazotrophic) microorganism is a key process affecting ecosystem functioning in soils. Understanding drivers affecting diazotrophic community assemblages and activities may lead to management practices to increase primary production and/or environmental sustainability. We used PCR-DGGE to determine the fundamental relationships between diazotrophic community structure and in a wide range of soils across southern Australia. In addition qPCR, RT-qPCR and N2-fixation (acetylene reduction) were used to investigate factors influencing gene abundance, expression and processes in similar soils with different agricultural inputs. Across 22 soils, the structural composition of the nifH community was significantly influenced by site (ANOSIM R = 0.876; P = 0.001). The effects of management practices were evident, and often larger than between-soil differences, but were only present at some sites. Differences in nifH communities between sites correlated to particulate organic carbon (POC; measured by mid-infrared spectroscopy) content of the soils (BIO-ENV test; ρ = 0.502; P = 0.001), but not other factors including total soil C. In 3 soils from the Murrumbidgee irrigation region of NSW, intensification of the farming systems was associated with increasing N2-fixation (P < 0.05), except where rice was cultivated. N2-fixation correlated either with nifH abundance or gene expression in soils, but not both. Our data shows that soil C is closely linked to diazotrophic ecology. Principally, the amount of C entering the soil system is directly related to the abundance and N2-fixation activity of free-living bacteria. However, we also show that C in the POC pool has associative links to the genetic diversity of the soil diazotroph community. Given the importance of diversity and abundance of functional organisms in supporting ecosystem processes, we suggest that soil C inputs should be considered for both qualitative and quantitative properties when considering impacts on diazotrophic bacterial ecology.DOI:10.1071/AR9830725URL [本文引用: 3]
DOI:10.1111/aec.2010.35.issue-2URL [本文引用: 1]
DOI:10.1007/s10021-009-9290-0URL [本文引用: 1]
Humid tropical forests are often characterized by large nitrogen (N) pools, and are known to have large potential N losses. Although rarely measured, tropical forests likely maintain considerable biological N fixation (BNF) to balance N losses. We estimated inputs of N via BNF by free-living microbes for two tropical forests in Puerto Rico, and assessed the response to increased N availability using an on-going N fertilization experiment. Nitrogenase activity was measured across forest strata, including the soil, forest floor, mosses, canopy epiphylls, and lichens using acetylene (C2H2) reduction assays. BNF varied significantly among ecosystem compartments in both forests. Mosses had the highest rates of nitrogenase activity per gram of sample, with 11±6nmol C2H2 reduced/g dry weight/h (mean±SE) in a lower elevation forest, and 6±1nmol C2H2/g/h in an upper elevation forest. We calculated potential N fluxes via BNF to each forest compartment using surveys of standing stocks. Soils and mosses provided the largest potential inputs of N via BNF to these ecosystems. Summing all components, total background BNF inputs were 120±29μg N/m2/h in the lower elevation forest, and 95±15μg N/m2/h in the upper elevation forest, with added N significantly suppressing BNF in soils and forest floor. Moisture content was significantly positively correlated with BNF rates for soils and the forest floor. We conclude that BNF is an active biological process across forest strata for these tropical forests, and is likely to be sensitive to increases in N deposition in tropical regions.
DOI:10.1111/j.1574-6941.2006.00235.xURLPMID:17116166 [本文引用: 1]
The effects of agronomic management practices on the soil microbial community were investigated in a maize production system in New South Wales, Australia. The site has been intensively studied to measure the impact of stubble management and N-fertilizer application on greenhouse gas emissions (CO(2) and N(2)O), N-cycling, pathology, soil structure and yield. As all of these endpoints can be regulated by microbial processes, the microbiology of the system was examined. Soil samples were taken after a winter fallow period and the diversity of the bacterial and fungal communities was measured using PCR-denaturing gradient gel electrophoresis. Stubble and N shifted the structure of bacterial and fungal communities with the primary driver being stubble addition on the fungal community structure (P<0.05 for all effects). Changes in C, N (total and NO(3)), K and Na, were correlated (P<0.05) with variation in the microbial community structure. Quantitative PCR showed that nifH (nitrogen fixation) and napA (denitrification) gene abundance increased upon stubble retention, whereas amoA gene numbers were increased by N addition. These results showed that the management of both stubble and N have significant and long-term impacts on the size and structure of the soil microbial community at phylogenetic and functional levels.
DOI:10.1071/SR08126URL
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DOI:10.1371/journal.pone.0042149URLPMID:22848735 [本文引用: 1]
The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples.
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DOI:10.1371/journal.pone.0110335URLPMID:25329300 [本文引用: 1]
We report on the contamination of commercial 15-nitrogen (15N) N2 gas stocks with 15N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. 15N2 gas is used to estimate N2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled 15N2 into organic matter. However, the microbial assimilation of bioavailable 15N-labeled N2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N2 fixation rates. 15N2 gas procured from three major suppliers was analyzed for the presence of these 15N-contaminants. Substantial concentrations of 15N-contaminants were detected in four Sigma-Aldrich 15N2 lecture bottles from two discrete batch syntheses. Per mole of 15N2 gas, 34 to 1900 micromoles of 15N-ammonium, 1.8 to 420 micromoles of 15N-nitrate/nitrite, and >/=21 micromoles of 15N-nitrous oxide were detected. One 15N2 lecture bottle from Campro Scientific contained >/=11 micromoles of 15N-nitrous oxide per mole of 15N2 gas, and no detected 15N-nitrate/nitrite at the given experimental 15N2 tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained >/=0.81 micromoles 15N-nitrous oxide per mole 15N2, and trace concentrations of 15N-ammonium and 15N-nitrate/nitrite. 15N2 gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the 15N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N2 fixation assays suggests that the degree of detected 15N-ammonium contamination could yield inferred N2 fixation rates ranging from undetectable, <0.01 nmoles N L(-1) d(-1), to 530 nmoles N L(-1) d(-1), contingent on experimental conditions. These rates are comparable to, or greater than, N2 fixation rates commonly detected in field assays. These results indicate that past reports of N2 fixation should be interpreted with caution, and demonstrate that the purity of commercial 15N2 gas must be ensured prior to use in future N2 fixation rate determinations.
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DOI:10.1128/AEM.01512-17URLPMID:29180374 [本文引用: 2]
The dinitrogenase reductase gene (nifH) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58 degrees C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field.IMPORTANCE Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.
DOI:10.3389/fmicb.2015.00731URLPMID:26236305 [本文引用: 1]
High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.
DOI:10.1128/aem.69.12.7289-7297.2003URLPMID:14660378 [本文引用: 1]
Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjarvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjarvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and ANABAENA: The main microcystin producer in Lake Tuusulanjarvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of ANABAENA: Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.
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DOI:10.1093/database/baaa102URLPMID:33306802 [本文引用: 1]
NPBS (Natural Products & Biological Sources) database is a chemical data resource with relational data between natural products and biological sources, manually curated from literatures of natural product researches. The relational data link a specific species and all the natural products derived from it and contrarily link a specific natural product and all the biological sources. The biological sources cover diverse species of plant, bacterial, fungal and marine organisms; the natural molecules have proper chemical structure data and computable molecular properties and all the relational data have corresponding references. NPBS database provides a wider choice of biological sources and can be used for dereplication to prevent re-isolation and re-characterization of already known natural products. Database URL: http://www.organchem.csdb.cn/scdb/NPBS.
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DOI:10.1128/AEM.49.2.423-428.1985URLPMID:16346730 [本文引用: 1]
Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO(2)/C(2)H(2) ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by N(2) incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels.
DOI:10.1016/j.soilbio.2006.01.022URL [本文引用: 1]
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DOI:10.1016/S0038-0717(02)00233-XURL [本文引用: 1]
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